ABSTRACT: In the last decades, the high incidence of viruses transmitted by whiteflies has become a problem in the tomato fields, threatening, more recently, the potato crops. The present study carried out a survey of begomoviruses and criniviruses in tomato and potato crops, from 2015 to 2018, in the municipalities of Araucária, Campo do Tenente, Campo Largo, Contenda, Lapa, Faxinal, Morretes, Reserva, Castro, Palmeira and São Mateus do Sul, in Paraná State, Brazil. Total DNA and RNA from leaves were extracted and used as templates to detect, respectively, begomoviruses by PCR and criniviruses by RT-PCR. Out of 215 tomato samples, 14 from Faxinal were infected by crinivirus. The other tomato samples and 243 potato samples showed negative results for begomovirus and crinivirus. Results indicated a low incidence (6.5%) of crinivirus infecting tomato crops in Paraná State, and the nucleotide sequence of one amplified fragment shared 99.71% identity with tomato chlorotic virus (ToCV).

RESUMO: Nas últimas décadas, a alta incidência de vírus transmitidos por mosca-branca tornou-se um problema nos campos de tomateiros, ameaçando, mais recentemente, a cultura da batateira. O presente trabalho teve como objetivo realizar um levantamento de begomovírus e crinivírus em lavouras de tomateiro e batateira nos municípios de Araucária, Campo do Tenente, Campo Largo, Contenda, Lapa, Faxinal, Morretes, Reserva, Castro, Palmeira e São Mateus do Sul, no Estado do Paraná, Brasil, de 2015 à 2018. DNA e RNA totais de folhas foram extraídos e utilizados como molde para detectar begomovírus por PCR e crinivírus por RT-PCR. Das 215 amostras de tomateiros coletadas, 14 provenientes de Faxinal estavam infectadas por crinivírus. As demais amostras de tomateiro e as 243 amostras de batateira analisadas apresentaram resultados negativo para begomovírus e crinivírus. Os resultados indicaram baixa incidência (6,5%) de crinivírus infectando lavouras de tomateiros no Estado do Paraná e a sequência de nucleotídeos de um amplicon apresentou 99,71% de identidade com o crinivírus tomato chlorotic virus (ToCV).

ABSTRACT Sugarcane-associated nematodes (Saccharum spp.) can reduce productivity up to 50%. Through the survey, it was possible to identify the main nematodes that occur in a certain region as a tool for designing the best management and control strategies. The aim of this study was to characterize the population of nematodes associated with sugarcane in the North Central, North Pioneiro and Northwest mesoregion of the state of Paraná, Brazil, quantify the nematode genera associated with the crop and identify the species of Pratylenchus and Meloidogyne. A total amount of 89 soil and root composite samples were collected in nine municipalities. Nematodes were extracted and counted in a Peters counting chamber under an optical light microscope. Morphological description followed identification keys. Pratylenchus spp. were identified by morphological characteristics; Meloidogyne spp. were identified by morphological characteristics and isoenzyme electrophoresis. Twelve genera of nematodes associated with sugarcane were identified: Pratylenchus, Meloidogyne, Helicotylenchus, Xiphinema, Mesocriconema, Trichodorus, Aphelenchus, Hoplolaimus, Tylenchus, Tylenchorhynchus, Ditylenchus, and Paratrichodorus. The genera Pratylenchus and Meloidogyne were found with the highest frequencies in the roots. Among the species of Pratylenchus, P. zeae and P. brachyurus were found, with P. zeae being the most frequent. Among the Meloidogyne species, only M. javanica was found. These results are essential to aid decision making in the management of phytonematodes, mainly in the development of new control strategies and in directing genetic breeding programs for development of sugarcane cultivars for the Paraná state.

RESUMO: Neste trabalho onze genótipos de cana-de-açúcar foram classificados em relação à sua resistência à ferrugem marrom, e onze quanto à resistência à ferrugem alaranjada. Foi realizada a inoculação no “cartucho foliar” de colmos com 165 dias, no município de Paranavaí, PR, em 2017. A avaliação foi feita aos 30 dias após a inoculação, utilizando uma escala de notas. Dos genótipos avaliados para ferrugem marrom, quatro foram classificados como suscetíveis, seis como moderadamente suscetíveis e um apresentou resistência moderada. Para a ferrugem alaranjada, três genótipos foram classificados como suscetíveis, sete como moderadamente suscetíveis e um como moderadamente resistente. A avaliação e classificação da reação de genótipos de cana-de-açúcar às ferrugens é uma ferramenta importante que auxilia em ensaios preliminares, na escolha de genótipos promissores para fases mais avançadas dos programas de melhoramento além de fornecer informações aos produtores na seleção das cultivares que serão plantadas.

ABSTRACT: In this research eleven sugarcane genotypes were classified in relation to their resistance to brown rust, and eleven to their resistance to orange rust. Artificial inoculation was carried out in the leaf whorl of 165-day-old plants in the city of Paranavaí, Paraná State, Brazil, in 2017. The evaluation was performed 30 days after inoculation, using a rating scale. Among the genotypes tested for brown rust, four were classified as susceptible, six as moderately susceptible and one presented moderate resistance. For orange rust, three genotypes were classified as susceptible, seven as moderately susceptible and one as moderately resistant. The evaluation and classification of the reaction of sugarcane genotypes to the rusts is an important tool that assist in preliminary trials and selection of promising genotypes for more advanced stages of breeding programs and provides information to producers on the choice of cultivars to be planted.

ABSTRACT: Brevipalpus-transmitted viruses (BTV) are a taxonomically diverse group of plant viruses which severely affect a number of major crops. Members of the group can be sub-classified into cytoplasmic (BTV-C) or nuclear type (BTV-N) according to the accumulation sites of virions in the infected plant cells. Both types of BTV produce only local infections near the point of inoculation by viruliferous mites. Features of BTV-plant interactions such as the failure of systemic spread in their natural hosts are poorly understood. In this study we evaluated Arabidopsis thaliana, a model plant commonly used for the study of plant-virus interactions, as an alternative host for BTV. Infection of Arabidopsis with the BTV-N Coffee ringspot virus and Clerodendrum chlorotic spot virus, and the BTV-C Solanum violaefolium ringspot virus, were mediated by viruliferous Brevipalpus mites collected in the wild. Upon infestation, local lesions appeared in 7 to 10 days on leaves of, at least, 80 % of the assayed plants. Presence of viral particles and characteristic cytopathic effects were detected by transmission electron microscopy (TEM) and the viral identities confirmed by specific reverse-transcriptase polymerase chain reaction (RT-PCR) and further amplicon sequencing. The high infection rate and reproducibility of symptoms of the three different viruses assayed validate A. thaliana as a feasible alternative experimental host for BTV.

Citrus leprosis (CL) is a serious threat to the citrus industry, especially for sweet oranges. For a long time, Citrus spp. were considered the only susceptible hosts. However, other plant species were also found either experimentally or naturally to be susceptible to Citrus leprosis virus C (CiLV-C). To assess the experimental host range of CiLV-C, a large number of plant species were inoculated with Brevipalpus phoenicis, viruliferous to CiLV-C, under experimental conditions. Out of the 140 tested species (43 families), 59 species (24 families) developed localized chlorotic and/or necrotic lesions upon inoculation of leaves with viruliferous mites, and 40 species (18 families) of them yielded positive results for CiLV-C detection in at least one of the following assays: ELISA, RT-PCR, transmission electron microscopy and immunfluorescence. For those that developed lesions and yielded negative results in CiLV-C detection assays, the results may be attributed to the small number of lesions and their necrotic state with very little viral material. The fact that a considerable number of plant species are susceptible to the virus after mite inoculation brings up implications for the epidemiology, quarantine and evolution of the citrus leprosis pathosystem.

Citrus leprosis (CL) was first described in South America in the 1920's. It is considered similar to a disease first observed back to 1860 in Florida. It is a destructive disease characterized by localized lesions on the leaves, fruits and stems, which may lead to the death of the affected plant if left untreated. Around 1940, CL was demonstrated to be transmitted by Brevipalpus mites in Argentina. Because little information exists on the status of CL pathosystem in Argentina and Paraguay, a survey was made in several citrus growing areas of these countries from 2009 to 2011, to evaluate its presence and relevance as well as the identification of the virus and the mite vector. CL was found in most of the sweet orange and/or mandarin orchards in Paraguay (Departamentos de Boquerón, Concepción, San Pedro, Cordillera, Alto Paraná, Itapúa) and Argentina (Provincias de Misiones, Corrientes, Entre Ríos). Incidence was usually low. The causal virus was identified as Citrus leprosis virus C (CiLV-C) by RT-PCR, electron microscopy and immunofluorescence. In all the visited regions in Paraguay and the region of Montecarlo, Argentina, the mites collected in plants infected by CiLV-C were identified as B. phoenicis.

This work reports the in vitro expression of Citrus leprosis virus C (CiLV-C) putative coat protein (p29) and the production of a polyclonal antibody to be used as a tool for serological diagnosis of citrus leprosis. The ORF2/RNA1, corresponding to p29, was cloned in pET28a and transformed into Escherichia coli cells (BL21). Expression of p29 was induced in vitro and the protein was purified and used for immunization of rabbits to produce the polyclonal antibody. The anti p29 serum was shown to be highly specific to CiLV-C detection by immunological methods (Western blot, PTA-ELISA, tissue blot and in situ immunolocalization), without cross reaction with healthy citrus plants or other cytoplasmic and nuclear viruses transmitted by Brevipalpus mites. These results demonstrate that the antibody against CiLV-C p29 protein is highly specific for CiLV-C detection. In situ immunogold labeling assays on thin sections of sweet orange leaf cells infected by CiLV-C demonstrated that short, bacilliform particles present within cisternae of the endoplasmic reticulum were specifically labeled, confirming their viral nature. The dense cytoplasmic viroplasm was also heavily labeled indicating that it represents a site of p29 accumulation.

Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plant-vector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.

Em 2005 houve um relato da presença da leprose dos citros no Estado de Chiapas, ao sul do México, baseado em sintomatologia. Subsequentemente a doença foi constatada em pomares de laranjeiras doces no Estado de Tabasco, nas localidades de Huimanguillo e Cunduacan. Amostras de folhas com lesões lepróticas de laranjeiras Valencia ou Hamlin foram coletadas e posteriormente examinadas ao microscópio eletrônico e submetidas a ensaios de detecção molecular por RT-PCR usando primers específicos. Estudos ultraestruturais revelaram alterações citopatológicas típicas causadas pelo CiLV-C (partículas baciliformes curtas no lúmen do retículo endoplasmático e a presença de viroplasmas vacuolados e elétron-densos no citoplasma) e hipertrofia em algumas células do parênquima lacunoso. Ensaios de RT-PCR resultaram na amplificação, na maioria das amostras de folhas dessecadas, de fragmento de DNA de tamanho esperado. Ácaros Brevipalpus coletados nos pomares foram identificados como B. phoenicis. Ensaios de transmissão com este ácaro confirmaram seu papel como vetor do CiLV-C para laranja doce, mas não houve transmissão para limão, limão Volkameriana, citrange C35 e citrange Carrizo. Todos estes dados considerados em conjunto confirmam que CiLV-C está presente em Tabasco, México e que a leprose dos citros descrita em Chiapas é causada pelo CiLV-C.

Citrus leprosis was detected in sweet oranges in Chiapas, Mexico, in 2005 based on symptoms. The disease was soon after observed in sweet orange orchards at Huimanguillo and Cunduacan, state of Tabasco. Leaf samples of leprosis-affected Valencia or Hamlin sweet oranges were collected and subjected to ultrastructural examination and molecular detection of CiLV-C by RT-PCR. Cytopathic effects typical of CiLV-C infection as presence of short, baciliform particles in the endoplasmic reticulum cisternae and electron dense, vacuolated viroplasma in the cytoplasm, as well as hypertrophy of some spongy parenchyma cells were observed in the tissues of the leaf lesions. RT-PCR using specific primers for CiLV-C produced DNA fragments of the expected size. Brevipalpus mites were present in these orchards and identified as B. phoenicis, the known vector for CiLV-C. Transmission tests with these mites confirmed their role as leprosis vectors to sweet orange but not to lemon, Volkamerian lemon, C35 citrange and Carrizo citrange. These data confirm the presence of CiLV-C in Tabasco, Mexico and that the citrus leprosis found in Chiapas was caused by CiLV-C.

O objetivo deste trabalho foi a caracterização biológica e molecular de um begomovírus detectado em tomateiros em São Joaquim de Bicas, Minas Gerais, denominado TGV-[Bi2]. A caracterização biológica consistiu em teste de gama de hospedeiros, realizado por meio de inoculação via extrato foliar tamponado ou bombardeamento de partículas. O isolado TGV-[Bi2] infecta plantas das famílias Solanaceae e Amaranthaceae, inclusive espécies economicamente importantes como o pimentão, e algumas plantas daninhas como Datura stramonium e Nicotiana silvestris. A caracterização molecular consistiu na clonagem e seqüenciamento de seu genoma completo (DNA-A e DNA-B). A comparação de seqüências e análise filogenética indicaram que o TGV-[Bi2] constitui uma nova espécie de begomovírus, denominada Tomato yellow spot virus (ToYSV), filogeneticamente relacionado ao Sida mottle virus (SiMoV).

The objective of this work was the biological and molecular characterization of a begomovirus detected in São Joaquim de Bicas, Minas Gerais, Brazil, named TGV-[Bi2], by determining its host range, complete nucleotide sequence and phylogenetic relationships with other begomoviruses. Biological characterization consisted of a host range study using either sap inoculation or particle bombardment as inoculation methods. The yellow spot virus can infect plants in Solanaceae and Amaranthaceae, including economically importat crops as sweet pepper, and weeds as Datura stramonium and Nicotiana silvestris. For the molecular characterization, the full-length genome (DNA-A and DNA-B) was amplified, cloned and completely sequenced. Sequence comparisons and phylogenetic analyses indicated that TGV-[Bi2] constitutes a novel begomovirus species named Tomato yellow spot virus (ToYSV), closely related to Sida mottle virus (SiMoV).

A análise da variabilidade genética de geminivírus infetando tomateiros (Lycopersicon esculentum) na região Sudeste do Brasil foi realizada por meio do seqüenciamento direto de fragmentos de PCR amplificados com oligonucleotídeos universais para o componente A de begomovírus, seguido de análise filogenética. Amostras de tomateiro e de algumas plantas daninhas associadas, apresentando sintomas típicos de infecção por vírus, foram coletadas em sete municípios nos estados de Minas Gerais, Espírito Santo e Rio de Janeiro. Após extração de DNA total e PCR, verificou-se que 137 amostras estavam infetadas por begomovírus, de um total de 369 amostras coletadas. A análise filogenética indicou existir uma grande diversidade genética de begomovírus em tomateiros nas três regiões amostradas, com a predominância de uma espécie viral (Tomato chlorotic mottle virus, TCMV) na Zona Metalúrgica de MG, enquanto no RJ e ES predomina uma outra espécie não completamente caracterizada. A análise filogenética indicou ainda a presença de outras quatro possíveis novas espécies de begomovírus em tomateiro. O alto grau de diversidade genética encontrado sugere uma transferência recente de begomovirus a partir de hospedeiros silvestres para o tomateiro. O relacionamento filogenético encontrado entre os begomovirus de tomateiro e isolados virais obtidos a partir de plantas daninhas favorece essa hipótese.

The genetic diversity of begomovirus isolates from tomato (Lycopersicon esculentum) fields in the Southeastern region of Brazil was analyzed by direct sequencing of PCR fragments amplified by using universal oligonucleotides for the begomovirus DNA-A, and subsequent computer-aided phylogenetic analysis. Samples of tomato plants and associated weeds showing typical symptoms of virus infection were collected at seven locations in the states of Minas Gerais, Espírito Santo and Rio de Janeiro. A total of 137 out of 369 samples were infected with a begomovirus based on PCR analysis. Phylogenetic analysis indicated a high degree of genetic diversity among begomoviruses infecting tomatoes in the sampled area. One species (Tomato chlorotic mottle virus, TCMV) occurs predominantly in Minas Gerais, whereas in Rio de Janeiro and Espírito Santo a distinct species, not yet fully characterized, predominates. Phylogenetic analysis further indicates the presence of an additional four possible new species. This high degree of genetic diversity suggests a recent transfer of indigenous begomovirus from wild hosts into tomatoes. The close phylogenetic relationship verified between begomovirus infecting tomato and associated weeds favors this hypothesis.